Nuclear Maturation Research Articles

Effect of Liquid Marble 3D Culture System on In Vitro Maturation and Embryo Development of Prepubertal Goat Oocytes

Suboptimal culture conditions during in vitro maturation (IVM) affect oocyte developmental competence and the viability of the resulting embryo. Three-dimensional (3D) culture systems provide a more biologically appropriate environment compared to traditional two-dimensional (2D) cultures. The aim of this study was to evaluate the effect of liquid marble (LM) microbioreactors as a 3D culture system on IVM and the subsequent embryo development of prepubertal goat oocytes. The cumulus–oocyte complexes (COCs) recovered from prepubertal goat ovaries underwent IVM in drops under oil (the 2D system and the control group) and in the 3D LM system (the LM group). After IVM, oocytes were parthenogenetically activated and cultured until the blastocyst stage. The control and LM groups showed similar rates of nuclear maturation (52.17% and 44.12%) and blastocyst formation (10.64% and 10.10%). Reactive oxygen species and glutathione levels and the density of transzonal projections (TZPs) in oocytes did not differ between groups. The LM system increased mitochondrial activity and modified the organization of these organelles in the oocyte cytoplasm compared to the control group. The LM microbioreactor demonstrated the ability to improve the mitochondrial status of the oocytes and was not harmful for oocyte IVM and subsequent embryo development. Therefore, LM could be used as a 3D cost-effective culture system for the IVM of prepubertal goat oocytes.

In Vitro Toxicity of a DEHP and Cadmium Mixture on Sheep Cumulus-Oocyte Complexes.

Di-(2-ethylhexyl) phthalate (DEHP) and Cadmium (Cd) affect female reproduction. To date, toxicological research has focused on the effects of individual contaminants, whereas living beings are exposed to mixtures. This study analyzed the effects of a DEHP/Cd mixture on nuclear and cytoplasmic maturation of sheep cumulus-oocyte complexes (COCs) compared with single compounds. COCs recovered from slaughterhouses-derived sheep ovaries were in vitro exposed to 0.5 μM DEHP, 0.1 μM Cd, or DEHP/Cd mixture at the same concentrations during 24 h of in vitro maturation (IVM). After IVM, oocyte nuclear chromatin configuration was evaluated, and bioenergetic/oxidative parameters were assessed on expanded cumulus cells (CCs) and matured oocytes (chi-square test and one-way ANOVA; p < 0.05). Under examined conditions, oocyte nuclear maturation was never impaired. However, COC bioenergetics was affected with stronger effects for the mixture than single compounds. Indeed, the percentages of matured oocytes with healthy mitochondrial distribution patterns were reduced (p < 0.001 and p < 0.05 for mixture and single compounds, respectively). Oocyte mitochondrial membrane potential, intracellular ROS levels, and mitochondria/ROS co-localization were reduced, with the same significance level, in all contaminated conditions. CCs displayed increased ROS levels only upon mixture exposure (p < 0.001). In conclusion, in vitro exposure to the DEHP/Cd mixture affected COC quality in the sheep to a greater extent than separate compounds.

Media Supplementation With Gamma-Oryzanol Improves the Outcome of Ovine Oocyte Maturation In Vitro.

The process of maturing ovine oocyte in vitro has not yet been raised with acceptable results. This study was designed to evaluate the γ-oryzanol effect as a supplement of maturation media on the development of ovine oocytes to blastocyst. Aspirated from ovine ovaries, morphologically normal cumulus-oocyte complexes (COCs) were matured in media supplemented with or without 5µM γ-oryzanol. Matured oocytes were divided into two parts: one evaluated for their nuclear maturation, the level of GSH and ROS, mitochondrial membrane potential (MMP) and the pattern of transcription in oocytes and respective cumulus cells (CCs), and another subjected to fertilisation and culture to assess the development of oocytes to the blastocyst. γ-Oryzanol improved the proportion of cleaved embryos and total blastocysts in the treated group, which was linked to improved MMP, higher levels of intracellular GSH and lower levels of ROS. A lower proportion of MI and GVBD was recorded for treated oocytes in comparison with control, although the proportion of MII oocytes was not different between groups. The treated oocytes and CCs showed downregulation of genes related to apoptosis (BAX and CASP-9) and upregulation of genes related to antioxidative status (NRF2, CAT and SOD). In conclusion, our results demonstrated the improved developmental outcome of supplemented oocytes so that the antioxidant response and higher enzymatic activity were maintained, and the generation of ROS was turned off; therefore, a novel alternative for counteracting oxidative stress in ovine oocytes undergoing maturation was offered by γ-oryzanol through an antioxidative pathway.

197 Influence of cumulus cell morphology on nuclear and cytoplasmic maturation markers of equine oocytes

197 Influence of cumulus cell morphology on nuclear and cytoplasmic maturation markers of equine oocytes

188 Influence of duration of in vitro maturation in relation to COC morphology on nuclear and cytoplasmic maturation of wood bison oocytes

188 Influence of duration of in vitro maturation in relation to COC morphology on nuclear and cytoplasmic maturation of wood bison oocytes

New aspect on the regulation of in vitro oocyte maturation: role of the obesity, neuropeptides and adipokines.

Oocyte quality determinants and nuclear and cytoplasmic maturation establish essential processes for fertilization and further development of the conceptus. Moreover, female fertility is strongly dependent on the metabolic status of the organism. Numerous sources indicate that obesity impairs ovarian function including oocyte physiology by inhibiting nuclear maturation, stimulating lipotoxicity and inflammation, enablingcumulus cells apoptosis, promotingreactive oxygen species formation and ultimately imposing pathogenic effects on mitochondria leading to infertility. Whereas, the number of overweight and obese individuals has reached alarming levels over the past decades, what is more, by 2030, the prevalence of overweight and obesity might reach 65.3% in adults in China and 78% in the USA. Thus, relationships between reproduction and metabolism are beingintensively studied to prevent obesity-induced infertility. The metabolic markers of oocyte condition and function are adipokines and neuropeptides, which regulate food intake, lipid and glucose metabolism, insulin resistance and impartsignificant influences on reproduction. Thus, in this review, we focus on interrelationships between obesity, oocyte maturation and the role of selected neuropeptides and adipokines including leptin, adiponectin, kisspeptin, nesfatin-1, phoenixin, visfatin, chemerin and vaspin.

Genetic Abnormalities of Oocyte Maturation: Mechanisms and Clinical Implications.

Genetic anomalies in oocyte maturation present significant fertility and embryonic development challenges. This review explores the intricate mechanisms of nuclear and cytoplasmic maturation, emphasizing the genetic and molecular factors contributing to oocyte quality and competence. Chromosomal mutations, errors in segregation, genetic mutations in signaling pathways and meiosis-related genes, and epigenetic alterations are discussed as critical contributors to oocyte maturation defects. The role of mitochondrial defects, maternal mRNA dysregulation, and critical proteins such as NLRP14 and BMP6 are highlighted. Understanding these genetic factors is crucial for improving diagnostic approaches and therapeutic interventions in reproductive medicine, particularly for couples encountering recurrent in vitro fertilization failures. This review will explore how specific genetic mutations impact fertility treatments and reproductive success by examining the intricate oocyte maturation process. We will focus on genetic abnormalities that may disrupt the oocyte maturation pathway, discussing the underlying mechanisms involved and considering their potential clinical implications for enhancing fertility outcomes.

Melatonin ameliorates the toxic effects of 2,6-Dichloro-1,4-benzoquinone on mouse oocytes by restoring subcellular structures

2,6-Dichloro-1,4-benzoquinone (2,6-DCBQ) is a disinfection by-product (DBP) formed during the disinfection of drinking water. Due to its frequent detection and high concentrations, it has garnered significant attention. However, the effects of 2,6-DCBQ on oocyte meiosis remain poorly understood. In this study, we demonstrate that 2,6-DCBQ exposure disrupts nuclear maturation in oocytes by damaging the spindle and chromosome structure. Additionally, exposure to 2,6-DCBQ impairs cytoplasmic maturation by altering actin dynamics, disrupting cortical granule distribution, and compromising the function of key organelles, including the mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. Supplementing melatonin during meiotic maturation reverses these effects, enhancing organelle function, reducing reactive oxygen species (ROS) levels, alleviating DNA damage, and inhibiting apoptosis. Together, these findings show that 2,6-DCBQ causes organelle dysfunction and meiotic disruption in oocytes, while melatonin provides protective effects against these disruptions during meiotic maturation.

Pyrroloquinoline-quinone supplementation restores ovarian function and oocyte quality in a mouse model of advanced maternal age.

Natural ovarian aging is one of the major causes for declining fertility in female animals, which has become an insurmountable issue in human reproduction clinics and assisted reproductive technology (ART) procedures. Nevertheless, the molecular basis of oocyte aging remains poorly understood, and feasible improvement strategies are unavailable. In the present study in vivo supplementation of pyrroloquinoline-quinone (PQQ) effectively elevated the fecundity of reproductively aged mice by balancing hormonal secretion, harmonizing the estrus cycle, and eliminating ovarian fibrosis. Moreover, oocyte quality also increased in aged mice after PQQ administration from various aspects, including nuclear and cytoplasmic maturation competency, fertilization capacity and pre-implantation embryonic development potential. Transcriptomic analysis identified target pathways that might mediate PQQ's effects in aged oocytes. Specifically, it was demonstrated that PQQ supplementation restored the mitochondrial dynamics and lysosomal function to remove excessive reactive oxygen species (ROS) and suppress apoptosis in aged oocytes. Jointly, these findings demonstrate PQQ administration is an efficacious method to restore the compromised ovary function and damaged oocyte quality in reproductively aged mice, which might be a potential clinical therapy for women of advanced maternal age with infertility.

Oocyte holding and in vitro maturation duration between 28 and 34 hours do not affect equine OPU-ICSI outcomes

Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in three different time windows using quartiles, with 465 oocytes (22.0 %) between 28 and 30 h (first quartile), 1078 oocytes (51.0 %) >30 and 31.7 h (second and third quartiles), and 571 oocytes (27.0 %) >31.7 and 34 h (fourth quartile). Using logistic regression models, the effect of duration of IVM with and without holding was tested on nuclear maturation, cleavage, blastocyst, and pregnancy rates. The three IVM intervals did not show differences in nuclear maturation (respectively 64.5 ± 0.48 %, 65.7 ± 0.47 %, and 67.3 ± 0.47 %), cleavage (respectively 59.7 ± 0.49 %, 58.5 ± 0.49 %, and 64.8 ± 0.48 %), blastocyst (respectively 17.5 ± 0.38 %, 19.0 ± 0.39 %, and 20.8 ± 0.41 %) nor pregnancy rates (respectively 65.4 ± 0.49 %, 70.3 ± 0.46 %, and 74.2 % ± 0.44) (P ≥ 0.38). Oocyte holding prior to IVM did not affect the results either (P ≥ 0.15). In conclusion, oocyte holding and IVM duration between 28 and 34h do not significantly affect outcomes, allowing flexibility in the planning of clinical OPU-ICSI in horses.

Oocyte competence develops: nuclear maturation synchronously with cytoplasm maturation.

Human oocyte maturation is a lengthy process that takes place over the course of which oocytes gain the inherent ability to support the next developmental stages in a progressive manner. This process includes intricate and distinct events related to nuclear and cytoplasmic maturation. Nuclear maturation includes mostly chromosome segregation, whereas rearrangement of organelles, storage of mRNAs and transcription factors occur during cytoplasmic maturation.Human oocyte maturation, both in vivo and in vitro, occurs through a process that is not yet fully understood. However, it is believed that the second messenger, cyclic adenosine monophosphate (cAMP), plays a pivotal role in the upkeep of the meiotic blocking of the human oocyte. Relatively high levels of cAMP in the human oocyte are required to maintain meiosis blocked, whereas lower levels of cAMP in the oocyte enable meiosis to resume. Oocyte cAMP concentration is controlled by a balance between adenylate cyclase and phosphodiesterases, the enzymes responsible for cAMP generation and breakdown.In addition to nuclear maturation, the female gamete requires a number of complicated structural and biochemical modifications in the cytoplasmic compartment to be able to fertilize normally. According to ultrastructural studies, during the transition from the germinal vesicle stage to metaphase II (MII), several organelles reorganize their positions. The cytoskeletal microfilaments and microtubules found in the cytoplasm facilitate these movements and regulate chromosomal segregation.The aim of this review is to focus on the nuclear and cytoplasmic maturation by investigating the changes that take place in the process of oocytes being competent for development.

Cumulus cell DNA damage linked to fertilization success in females with an ovulatory dysfunction phenotype.

Intracytoplasmic sperm injection (ICSI) is a widely used technique in fertility centers. ICSI success depends on both nuclear and cytoplasmic oocyte maturation. Cumulus cells, which surround the oocytes, play a pivotal role in oocyte competence. However, the significance of DNA damage in cumulus cells as a marker of fertilization success remains largely unexplored. This study aims to investigate the relationship between DNA damage in cumulus cells of females undergoing ICSI, and oocyte competence, with a focus on in vitro fertilization (IVF) outcomes. We employed the alkaline comet assay to assess DNA damage levels (%TDNA) in cumulus cells and whole blood from 22 potentially fertile females and 35 infertile females, including 20 with an ovulatory disfunction phenotype. Our results revealed significant differences between the levels of %TDNA in cumulus cells and blood. Females with an ovulatory dysfunction phenotype exhibited higher levels of %TDNA in cumulus cells compared to potentially fertile females. Additionally, within the group of females with ovulatory dysfunction, a significant correlation was observed between %TDNA levels and the number of oocytes with two pronuclei. Our findings suggest that blood does not accurately reflect DNA damage in cumulus cells, which was correlated with the fertilization success in females with ovulatory dysfunction. High levels of %TDNA in cumulus cells were associated with a higher likelihood of successful fertilization. Moreover, our results imply that low levels of %TDNA may be linked to oocytes that fail to complete maturation and, consequently, do not fertilize (oocytes with zero pronuclei). Further research with larger cohorts is necessary to validate these findings and to explore potential applications in female fertility. However, our study provides evidence that DNA damage in cumulus cells may serve as a valuable biomarker for predicting fertilization success and oocyte competence.

Abnormalities of Oocyte Maturation: Mechanisms and Implications.

The elucidation of oocyte maturation mechanisms is paramount for advancing embryo development within the scope of assisted reproductive technologies (ART). Both cytoplasmic and nuclear maturation represent intricate processes governed by tightly regulated cellular pathways, which are essential for ensuring the oocyte's competence for fertilization and subsequent embryogenesis. A comprehensive grasp of these mechanisms is vital, as the maturation stage of the oocyte significantly influences chromosomal integrity, spindle formation, and its ability to support the initial stages of embryonic development. By leveraging this knowledge, we can enhance in vitro fertilization (IVF) protocols, refining ovarian stimulation regimens and culture conditions to improve oocyte quality. This, in turn, has the potential to boost pregnancy rates and outcomes. Further research in this area will contribute to the development of novel interventions that aim to increase the efficacy of preimplantation embryonic development, offering new opportunities for individuals undergoing fertility treatments.

Associations of Ureaplasma urealyticum infection with male infertility and intrauterine insemination outcomes.

Ureaplasma urealyticum (UU) is one of the most commonly occurring pathogens associated with genital tract infections in infertile males, but the impact of seminal UU infection in semen on intrauterine insemination (IUI) outcomes is poorly understood. We collected data from 245 infertile couples who underwent IUI at The First Affiliated Hospital of USTC (Hefei, China) between January 2021 and January 2023. The subjects were classified into two groups according to their UU infection status: the UU-positive group and the UU-negative group. We compared semen parameters, pregnancy outcomes, and neonatal birth outcomes to investigate the impact of UU infection on IUI outcomes. There were no significantly statistical differences in various semen parameters, including semen volume, sperm concentration, total and progressive motility, sperm morphology, leukocyte count, the presence of anti-sperm antibody, and sperm DNA fragmentation index (DFI), between the UU-positive and UU-negative groups of male infertile patients (all P > 0.05). However, the high DNA stainability (HDS) status of sperm differed between the UU-positive and UU-negative groups, suggesting that seminal UU infection may affect sperm nuclear maturation (P = 0.04). Additionally, there were no significant differences in pregnancy or neonatal birth outcomes between the two groups (all P > 0.05). These results suggest that IUI remains a viable and cost-effective option for infertile couples with UU infection who are facing infertility issues.

Increased in vitro production of bovine embryos resulting from oocyte maturation in the presence of triciribine, a specific inhibitor of AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX). COCs were fertilized in vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1 μM of triciribine were stained at 3, 6, and 9 h of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5 % significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P < 0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P < 0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P < 0.05). On the other hand, when COCs were matured in the presence of 1 μM, there was an increase in the blastocyst rate (P < 0.05), but without altering the timing of meiosis resumption (P < 0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1 μM of triciribine increases the in vitro production of bovine embryos.

RNA surveillance by the RNA helicase MTR4 determines volume of mouse oocytes.

Oocytes are the largest cell type in multicellular animals. Here, we show that mRNA transporter 4 (MTR4) is indispensable for oocyte growth and functions as part of the RNA surveillance mechanism, which is responsible for nuclear waste RNA clearance. MTR4 ensures the normal post-transcriptional processing of maternal RNAs, their nuclear export to the cytoplasm, and the accumulation of properly processed transcripts. Oocytes with Mtr4 knockout fail to accumulate sufficient and normal transcripts in the cytoplasm and cannot grow to normal sizes. MTR4-dependent RNA surveillance has a previously unrecognized function in maintaining a stable nuclear environment for the establishment of non-canonical histone H3 lysine-4 trimethylation and chromatin reorganization, which is necessary to form a nucleolus-like structure in oocytes. In conclusion, MTR4-dependent RNA surveillance activity is a checkpoint that allows oocytes to grow to a normal size, undergo nuclear and cytoplasmic maturation, and acquire developmental competence.

A transcription-independent role for HIF-1α in modulating microprocessor assembly.

Microprocessor is an essential nuclear complex responsible for the initial RNase-mediated cleavage of primary miRNA, which is a tightly controlled maturation process that requires the proper assembly of Drosha and DGCR8. Unlike previously identified mechanisms directly targeting the enzymatic subunit Drosha, current knowledge about the biological ways of controlling miRNA nuclear maturation through DGCR8 is less addressed. In this study, we unveiled that the microprocessor assembly is governed by a master gene regulator HIF-1α irrespective of its canonical transcriptional activity. First, a widespread protein binding of HIF-1α with DGCR8 instead of Drosha was observed in response to biological stimulations. Similar protein interactions between their corresponding orthologues in model organisms were also observed. After dissecting the essential protein domains, we noticed that HIF-1α suppresses microprocessor assembly via binding to DGCR8. Furthermore, our results showed that HIF-1α hijacks monomeric DGCR8 thus reducing its dimer formation prior to microprocessor assembly, and consequently, the suppressed microprocessor formation and nuclear processing of primary miRNA were demonstrated. In conclusion, here we unveiled the mechanism of how microprocessor assembly is regulated by HIF-1α, which not only demonstrates a non-transcriptional function of nuclear HIF-1α but also provides new molecular insights into the regulation of microprocessor assembly through DGCR8.

Defenders of the Transcriptome: Guard Protein-Mediated mRNA Quality Control in Saccharomyces cerevisiae.

Cell survival depends on precise gene expression, which is controlled sequentially. The guard proteins surveil mRNAs from their synthesis in the nucleus to their translation in the cytoplasm. Although the proteins within this group share many similarities, they play distinct roles in controlling nuclear mRNA maturation and cytoplasmic translation by supporting the degradation of faulty transcripts. Notably, this group is continuously expanding, currently including the RNA-binding proteins Npl3, Gbp2, Hrb1, Hrp1, and Nab2 in Saccharomyces cerevisiae. Some of the human serine-arginine (SR) splicing factors (SRSFs) show remarkable similarities to the yeast guard proteins and may be considered as functional homologues. Here, we provide a comprehensive summary of their crucial mRNA surveillance functions and their implications for cellular health.

Melatonin facilitates oocyte growth in goats and mice through increased nutrient reserves and enhanced mitochondrial function.

Oogenesis involves two phases: initial volumetric growth driven by nutrient accumulation and subsequent nuclear maturation. While melatonin (MLT) has been employed as a supplement to enhance the quality of fully grown oocytes during nuclear maturation phase, its impact on oocyte growth remains poorly studied. Here, we provide invivo evidence demonstrating that follicle-stimulating hormone increases MLT content in ovary. Administration of MLT improves oocyte growth and quality in mice and goats by enhancing nutrient reserves and mitochondrial function. Conversely, MLT-deficient mice have smaller oocytes and dysfunctional mitochondria. Exploring the clinical implications of MLT in promoting oocyte growth, we observe that a brief 2-day MLT treatment enhances oocyte quality and reproductive performance in older mice. These findings highlight the role of MLT in regulating oocyte growth and provide a specific treatment window for optimizing oocyte quality and reproductive performance in female animals.

VPS34 Governs Oocyte Developmental Competence by Regulating Mito/Autophagy: A Novel Insight into the Significance of RAB7 Activity and Its Subcellular Location.

Asynchronous nuclear and cytoplasmic maturation in human oocytes is believed to cause morphological anomalies after controlled ovarian hyperstimulation. Vacuolar protein sorting 34 (VPS34) is renowned for its pivotal role in regulating autophagy and endocytic trafficking. To investigate its impact on oocyte development, oocyte-specific knockout mice (ZcKO) are generated, and these mice are completely found infertile, with embryonic development halted at 2- to 4-cell stage. This infertility is related with a disruption on autophagic/mitophagic flux in ZcKO oocytes, leading to subsequent failure of zygotic genome activation (ZGA) in derived 2-cell embryos. The findings further elucidated the regulation of VPS34 on the activity and subcellular translocation of RAS-related GTP-binding protein 7 (RAB7), which is critical not only for the maturation of late endosomes and lysosomes, but also for initiating mitophagy via retrograde trafficking. VPS34 binds directly with RAB7 and facilitates its activity conversion through TBC1 domain family member 5 (TBC1D5). Consistent with the cytoplasmic vacuolation observed in ZcKO oocytes, defects in multiple vesicle trafficking systems are also identified in vacuolated human oocytes. Furthermore, activating VPS34 with corynoxin B (CB) treatment improved oocyte quality in aged mice. Hence, VPS34 activation may represent a novel approach to enhance oocyte quality in human artificial reproduction.